Choleragen and A protomer catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide. The protein with NAD glycohydrolase activity migrated with choleragen on polyacrylamide gels and chromatographed with the A protomer on BioGel P60 columns. NAD hydrolysis was increased in acetate and phosphate buffers, and enhanced greater than 10-fold by dithiothreitol in high concentration. NADP was hydrolyzed slowly by choleragen. Analogues of NAD inhibited the glycohydrolase activity, adenine being the most effective. Formation of nicotinamide from NaD was enhanced by L-arginine and guanidine; L-citrulline was ineffective. The product formed after incubation of (adenine-U-14C)NAD and (3H)-L-arginine with choleragen contained 14C and H in the 1:1 ratio expected of ADP-ribose-L-arginine. These results are consistent with choleragen possessing ADP-ribosyltransferase activity with arginine serving as acceptor. Activation of adenylate cyclase may involve the ADP-ribosylation of an arginine in an acceptor protein, which is either the cyclase itself or is involved in cyclase activation. BIBLIOGRAPHIC REFERENCES: Moss, J., Richards, R.L., Alving, C. R., and Fishman, P.H. Effect of the A and B protomers of choleragen on release of trapped glucose from liposomes containing or lacking ganglioside GM1. J. Biol. Chem. Moss, J., Osborne, J.C., Jr., Fishman, P.H., Brewer, H.B., Jr., Vaughan, M., and Brady, R.O. Effect of gangliosides and substrate analogues on the hydrolysis of nicotinamide adenine dinucleotide by chloragen. Proc. Natl. Acad. Sci. USA 74:74-78, 1977.